Glycomimetic inhibitors of the PA-IL lectin, PA-IIL lectin or both the lectins from pseudomonas

ABSTRACT

Compositions and methods are provided related to  Pseudomonas  bacteria. The compositions and methods may be used for diagnosis and therapy of medical conditions involving infection with  Pseudomonas  bacteria. Such infections include  Pseudomonas aeruginosa  in the lungs of patients with cystic fibrosis. A compound useful in the present methods may be used in combination with a therapeutic agent or may be linked to a therapeutic agent.  Pseudomonas  bacteria may be inhibited by blocking colonization, inhibiting virulence factors, arresting growth or killing the bacteria.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/501,464, filed Aug. 8, 2006, now issued as U.S. Pat. No. 7,517,980 onApr. 14, 2009; which application claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Patent Application No. 60/706,546 filed Aug.9, 2005 and U.S. Provisional Patent Application No. 60/810,190 filedJun. 1, 2006; which applications are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compounds, compositions andmethods for the diagnosis and therapy of diseases in warm-bloodedanimals (e.g., in humans) involving infections with and colonization byPseudomonas bacteria, including Pseudomonas aeruginosa in the lungs ofpatients with cystic fibrosis. The invention relates more particularlyto the use of one or more compounds selective for binding PA-IL and/orPA-IIL lectins of Pseudomonas bacteria. These compounds are useful fordiagnosis and/or therapeutic intervention of the colonization ofPseudomonas bacteria, or may be linked to an agent(s) to target andeffectively arrest or kill Pseudomonas bacteria.

2. Description of the Related Art

Pseudomonas infections occur in a variety of medical conditions and canbe life threatening. Pseudomonas is an opportunistic bacterium. Examplesof individuals at risk include cystic fibrosis patients, burn patients,and patients on ventilators. Cystic fibrosis is described below as arepresentative example of a medical condition which can involveinfection with Pseudomonas bacteria.

Cystic Fibrosis (CF) is the most common lethal genetic disease among theCaucasian population. CF is caused by mutations in the gene encoding thecystic fibrosis transmembrane conductance regulator (CFTR), which actsas a chloride channel. The genetic mutations of CFTR which alter ionmovements also affect the N-glycosylation of CFTR as well as other cellsurface molecules. All of the exocrine glands of the patients areaffected; however, the lungs are the primary site of morbidity andmortality. The general change in glycosylation is associated with anincrease in infectivity by Pseudomonas aeruginosa. The salivary andrespiratory mucins from CF patients also contain altered glycosylationpatterns.

The major cause of morbidity and mortality in CF patients is chroniclung colonization by the bacterium, Pseudomonas aeruginosa, whichresults in pronounced lung infection with a robust neutrophilicinflammatory response leading to destruction of the lungs and death.Colonization by P. aeruginosa initiates during the sessile phase of thebacteria in which virulence factors are secreted in concert. Twovirulence factors that bind carbohydrates are lectins. These lectins,known as PA-IL and PA-IIL, bind these oligosaccharide structures withhigh affinity and represent a potential molecular target to blockbacterial colonization. Patients that are never fully colonized by thebacteria maintain an excellent long-term prognosis. Due to thedifficulties in the current approaches in the art for prevention ofcolonization in an individual by Pseudomonas bacteria, there is a needfor improved compounds, compositions and methods.

BRIEF SUMMARY OF THE INVENTION

Briefly stated, this invention provides compounds, compositions andmethods for utilizing both the PA-IIL and PA-IL lectins, or either onealone, expressed by Pseudomonas bacteria for the detection ofPseudomonas bacteria and the diagnosis and therapy of disease involvingPseudomonas bacteria, including human disease. For example, compounds ofthe present invention that have high affinity binding to the PA-IILlectin, the PA-IL lectin or both lectins from P. aeruginosa will have abeneficial therapeutic effect on CF patients. Furthermore, thesecompounds may be administered in combination therapy with antibiotics ormay be conjugated, for example, with antibiotics to increase theefficacy and lower the dose, thereby avoiding well known deleteriousside effects of many antibiotics. Given that these binding sites arecrucial for the colonization and pathogenicity of the bacterium,mutations in this target to become resistant to this conjugate therapyshould result in non-pathogenic forms of the bacteria.

One embodiment of the present invention provides a compound orphysiologically acceptable salt thereof, having the formula:

wherein:

-   -   where —O— separating the two rings in the formula is in an alpha        or beta 1-3 linkage;    -   R¹=independently selected from OH, NHAc, 6′ sulfated GlcNAc, 6′        carboxylated GlcNAc, GalNAc, galactose linked by an O glycosidic        bond, a C glycosidic bond or an S glycosidic bond,        thiodigalactoside, 6′ sulfated galactose and 6′ carboxylated        galactose, with the proviso that three of the four R¹ are        independently selected from OH and NHAC and one R¹ is not OH or        NHAC;    -   R²=H, a fucose, a galactose, an arabinose, a fructose, a        mannose, cyclohexane, substituted cyclohexane, tetrahydropyran,        substituted tetrahydropyran, piperidine, substituted piperidine,        a polyol or substituted polyol;    -   R³=(CH₂)_(p), NH₂, —CH₂—OH, —CH₂        NH        _(q)X or

-   -    where m, n and q are independently selected from 0-1, p is        1-20, Z is N, O or S, and X is NH—C₁-C₈ alkyl, C₃-C₈ cycloalkyl,        substituted C₃-C₈ cycloalkyl, C₁-C₈ alkyl, C₆-C₁₄ aryl,        substituted C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, substituted C₁-C₁₄        heteroaryl, non-aryl C₁-C₁₄ heterocycle or substituted non-aryl        C₁-C₁₄ heterocycle, NHCH₂Ph, N(CH₂Ph)₂, NHSO₃Na, NHCO—C₆H₄—COOH        (ortho), NHCOPh, NHCO—C₆H₄—Cl (para), NHCO—C₆H₄—OMe (para),        NHCO—C₆H₄—NO₂ (para), NHCO—C₆H₄-Ph (para), NHCO—C₆H₃(OMe)₂        (meta, para), NHCO(2-naphthyl), NHCO—C₆H₄—OCH₂Ph (para),        N(CH₂Ph)COPh, NHCOCH₂CH₂Ph, NHCOCHPh₂, NHCOMe,        NHCO(cyclo-C₆H₁₁), NHSO₂—C₆H₄-Me (para), NHCONHEt, NHCONHPh,        NHCOOCH₂—C₆H₄—NO₂ (para), NHCOOCH₂(2-naphthyl), or NHCOOCH₂Ph;    -   R⁴=H, NHAc, —O-Lactose, substituted —O-Lactose, —O-Lactosamine,        substituted —O-Lactosamine, NHAc substituted with N-glycolyl,        polyethylene glycol or substituted polyethylene glycol; and    -   R⁵=H, NHAc, or NHAc substituted with N-glycolyl.        A compound or salt thereof of the present invention may be in        combination with a pharmaceutically acceptable carrier or        diluent.

In another embodiment, the present invention provides a conjugatecomprising a therapeutic agent linked to a compound as set forth above.

Another embodiment of the present invention provides a method ofinhibiting Pseudomonas bacteria infection in a warm-blooded animalcomprising administering to the animal in an amount effective to inhibitone or more lectins of the bacteria a compound comprising a compound orphysiologically acceptable salt thereof, having the formula:

wherein:

-   -   where —O— separating the two rings in the formula is in an alpha        or beta 1-3 linkage;    -   R¹=independently selected from OH, NHAc, 6′ sulfated GlcNAc, 6′        carboxylated GlcNAc, GalNAc, galactose linked by an O glycosidic        bond, a C glycosidic bond or an S glycosidic bond,        thiodigalactoside, 6′ sulfated galactose and 6′ carboxylated        galactose, with the proviso that three of the four R¹ are        independently selected from OH and NHAc and one R¹ is not OH or        NHAc;    -   R²=H, a fucose, a galactose, an arabinose, a fructose, a        mannose, cyclohexane, substituted cyclohexane, tetrahydropyran,        substituted tetrahydropyran, piperidine, substituted piperidine,        a polyol or substituted polyol;    -   R³=(CH₂)_(p), NH₂, —CH₂—OH, —CH₂        NH        _(q)X or

-   -    where m, n and q are independently selected from 0-1, p is        1-20, Z is N, O or S, and X is NH—C₁-C₈ alkyl, C₃-C₈ cycloalkyl,        substituted C₃-C₈ cycloalkyl, C₁-C₈ alkyl, C₆-C₁₄ aryl,        substituted C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, substituted C₁-C₁₄        heteroaryl, non-aryl C₁-C₁₄ heterocycle or substituted non-aryl        C₁-C₁₄ heterocycle, NHCH₂Ph, N(CH₂Ph)₂, NHSO₃Na, NHCO—C₆H₄—COOH        (ortho), NHCOPh, NHCO—C₆H₄—Cl (para), NHCO—C₆H₄—OMe (para),        NHCO—C₆H₄—NO₂ (para), NHCO—C₆H₄-Ph (para), NHCO—C₆H₃(OMe)₂        (meta, para), NHCO(2-naphthyl), NHCO—C₆H₄—OCH₂Ph (para),        N(CH₂Ph)COPh, NHCOCH₂CH₂Ph, NHCOCHPh₂, NHCOMe,        NHCO(cyclo-C₆H₁₁), NHSO₂—C₆H₄-Me (para), NHCONHEt, NHCONHPh,        NHCOOCH₂—C₆H₄—NO₂ (para), NHCOOCH₂(2-naphthyl), or NHCOOCH₂Ph;    -   R⁴=H, NHAc, —O-Lactose, substituted —O-Lactose, —O-Lactosamine,        substituted —O-Lactosamine, NHAc substituted with N-glycolyl,        polyethylene glycol or substituted polyethylene glycol; and    -   R⁵=H, NHAc, or NHAc substituted with N-glycolyl.

In another embodiment, the present invention provides a method ofdetecting Pseudomonas bacteria comprising contacting a sample with adiagnostic agent linked to a compound comprising a compound as set forthabove, under conditions sufficient for the compound to bind to thebacteria or its lectin products if present in the sample; and detectingthe agent present in the sample, wherein the presence of agent in thesample is indicative of the presence of Pseudomonas bacteria.

In another embodiment, the present invention provides a method ofimmobilizing Pseudomonas bacteria on a solid support comprisingcontacting, under conditions sufficient for binding, a sample containingPseudomonas bacteria with a compound comprising a compound as set forthabove that is immobilized on a solid support; and separating the samplefrom the solid support.

In other embodiments, the compounds and conjugates described herein maybe used in the preparation of a medicament for the inhibition ofPseudomonas bacteria.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the synthesis of a glycomimeticcompound.

FIG. 2A is a diagram illustrating the synthesis of a glycomimeticcompound containing a linker arm.

FIG. 2B is a diagram illustrating the conjugation of the compound ofFIG. 2A to the antibiotic tobramycin.

FIGS. 3A and 3B are diagrams illustrating the synthesis of glycomimeticcompounds.

FIG. 4 depicts the structures of three of the compounds (Compound A,Compound B, and Glycomimetic 1) used in one or more of the lectin assaysdescribed herein.

FIG. 5 graphically illustrates the inhibition of PA-IL lectin byGlycomimetic 1 (“Glycm 1”). PA-IL lectin is a galactose-binding lectinand is inhibited by galactose, Melibiose (Galα1-6Gal), and Glycomimetic1; but not by fucose, Compound A or Compound B.

FIG. 6 graphically illustrates inhibition of the PA-IIL Lectin. Thefucose binding lectin, PA-IIL is inhibited by fucose, Compound A andGlycomimetic 1; but not by galactose.

FIG. 7 shows the determination of IC₅₀ value for Glycomimetic 1 forinhibition of PA-IL. Glycomimetic 1 inhibits the galactose-bindinglectin, PA-IL about 4 to 5 times better than galactose; whereas, fucoseis inactive.

FIG. 8 shows the IC₅₀ determination of Glycomimetic 1 for inhibition ofPA-IIL. Glycomimetic 1 inhibits the fucose-binding lectin, PA-IIL about4 to 5 times better than fucose; while galactose is inactive.

FIG. 9A-9F illustrates the inhibition of PA-IIL binding to humanepithelial cells. Human buccal epithelial cells are incubated withbiotinylated PA-IIL lectin followed by detection of bound lectin withfluorescein-labeled streptavidin. Fluorescently labeled cells arequantified by fluorescent-activated cell sorting (FIG. 9B). Incubationof the PA-IIL lectin with fucose (FIG. 9D) or Glycomimetic 1 (FIG. 9Eand 9F) inhibits binding to the cell surface. Galactose (FIG. 9C) has noeffect.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides compounds andcompositions that bind Pseudomonas bacterial lectins (e.g., P.aeruginosa lectins) and may be used in the diagnosis and therapy ofdisease.

Glycomimetic Compounds

The term “Glycomimetic compound,” as used herein, refers to a compound(including physiologically acceptable salts thereof) that has highaffinity for the PA-IL lectin, PA-IIL lectin or both lectins fromPseudomonas bacteria. Embodiments of the structures of the Glycomimeticcompounds of this invention have the formula:

wherein:

-   -   where —O— separating the two rings in the formula is in an alpha        or beta 1-3 linkage;    -   R¹=independently selected from OH, NHAc, 6′ sulfated GlcNAc, 6′        carboxylated GlcNAc, GalNAc, galactose linked by an O glycosidic        bond, a C glycosidic bond or an S glycosidic bond,        thiodigalactoside, 6′ sulfated galactose and 6′ carboxylated        galactose;    -   R²=H, a fucose, a galactose, an arabinose, a fructose, a        mannose, cyclohexane, substituted cyclohexane, tetrahydropyran,        substituted tetrahydropyran, piperidine, substituted piperidine,        a polyol or substituted polyol;    -   R³=(CH₂)_(p), NH₂, —CH₂—OH, —CH₂        NH        _(q)X or

-   -    where m, n and q are independently selected from 0-1, p is        1-20, Z is N, O or S, and X is NH—C₁-C₈ alkyl, C₃-C₈ cycloalkyl,        substituted C₃-C₈ cycloalkyl, C₁-C₈ alkyl, C₆-C₁₄ aryl,        substituted C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, substituted C₁-C₁₄        heteroaryl, non-aryl C₁-C₁₄ heterocycle or substituted non-aryl        C₁-C₁₄ heterocycle, NHCH₂Ph, N(CH₂Ph)₂, NHSO₃Na, NHCO—C₆H₄—COOH        (ortho), NHCOPh, NHCO—C₆H₄—Cl (para), NHCO—C₆H₄-OMe (para),        NHCO—C₆H₄—NO₂ (para), NHCO—C₆H₄-Ph (para), NHCO—C₆H₃(OMe)₂        (meta, para), NHCO(2-naphthyl), NHCO—C₆H₄—OCH₂Ph (para),        N(CH₂Ph)COPh, NHCOCH₂CH₂Ph, NHCOCHPh₂, NHCOMe,        NHCO(cyclo-C₆H₁₁), NHSO₂—C₆H₄-Me (para), NHCONHEt, NHCONHPh,        NHCOOCH₂—C₆H₄—NO₂ (para), NHCOOCH₂(2-naphthyl), or NHCOOCH₂Ph;    -   R⁴=H, NHAC, —O-Lactose, substituted —O-Lactose, —O-Lactosamine,        substituted —O-Lactosamine, NHAC substituted with N-glycolyl,        polyethylene glycol or substituted polyethylene glycol; and    -   R⁵=H, NHAC, or NHAC substituted with N-glycolyl.        All compounds (or conjugates thereof) useful in the present        invention include physiologically acceptable salts thereof.

Glycomimetic compounds of the present invention include the formula setforth above with substituents R¹-R⁵. Where a substituent option (i.e.,atom or group) for R¹-R⁵ possesses a “-” this is to indicate the pointof attachment (to a ring for R^(1a), R^(1b), R^(1c) and R³-R⁵, to CH₂for R^(1d), and to O for R²), and does not represent CH₂ or CH₃. In theabove formula, there is an oxygen (—O—) linking the two rings depictedin the formula. The oxygen may be in an alpha 1-3 linkage or a beta 1-3linkage.

As used herein, a line to which no group is depicted represents the bondthat attaches the substituent to the structure depicted by the generalformula. As used herein, a “C₆-C₁₄ aryl” refers to an aromaticsubstituent with six to fourteen carbon atoms in one or multiple ringswhich may be separated by a bond or an alkyl group or be fused. As usedherein, a “C₁-C₁₄ heteroaryl” is similar to a “C₆-C₁₄ aryl,” except thearomatic substituent possesses at least one heteroatom (such as N, O orS) in place of a ring carbon. Examples of aryls and heteroaryls includephenyl, naphthyl, diphenyl, pyridinyl and pyrimidinyl.

R¹ is composed of R^(1a), R^(1b), R^(1c) and R^(1d), as depicted in theabove formula. R^(1a) is attached at carbon position 2. R^(1b) isattached at carbon position 3. R^(1c) is attached at carbon position 4.R^(1d) is attached at carbon position 6, which in turn is attached atcarbon position 5.

Examples of R¹ substituents include GalNAc, 6′ sulfated GlcNAc and 6′carboxylated GlcNAc. The abbreviation “GlcNAc” representsN-Acetylglucosamine and “GalNAc” represents N-Acetylgalactosamine. OtherR¹ substituents are OH, NHAc, galactose, thiodigalactoside, 6′ sulfatedgalactose and 6′ carboxylated galactose. Galactose is linked by an Oglycosidic bond, a C glycosidic bond or an S glycosidic bond. Where R¹as set forth with the above formula is galactose, 6′ sulfated galactoseor 6′ carboxylated galactose, R¹ in an embodiment is attached by analpha 1-3 linkage, but the linkage may be beta. In embodiments, only oneof the four R¹ is other than OH or NHAc (i.e., selected from one of theR¹ substituents listed other than OH or NHAc).

Examples of R² substituents include monosaccharides, such as fucose,galactose, arabinose, fructose or mannose. The monosaccharides possess aD- and an L-form. Such monosaccharides include L-fucose, L-galactose,D-arabinose, D-fructose and D-mannose. A monosaccharide of R² may bereplaced with a mimic of the monosaccharide. For example, amonosaccharide ring may be replaced with a cyclohexane, substitutedcyclohexane, tetrahydropyran, substituted tetrahydropyran, piperidine,substituted piperidine, a polyol or substituted polyol. Alternatively,or in addition to the replacement of a ring, substituents may be addedto a ring as replacement for, or in addition to, existing substituentsor both. For example, one or more hydroxyl groups may be replaced withalkoxy groups (such as methoxy, ethoxy, propoxy, etc.), halides (such asfluorine, chlorine, etc.), esters and amides. Similarly, for example,one or more hydrogens of cyclohexane, tetrahydropyran or piperidine maybe replaced with such groups (alkoxy, halide, ester and amide) toproduce a substituted cyclohexane, substituted tetrahydropyran orsubstituted piperidine, respectively.

Examples of R³ substituents include (CH₂)_(p) where p is 1-20, —CH₂—OH,NH₂, —CH₂—(NH)_(q)—X and —(CH₂)_(m)—NH—C(═O)—(Z)_(n)—X where m, n and qare independently selected from 0 and 1, and where X is NH—C₁-C₈ alkyl,C₃-C₈ cycloalkyl, substituted C₃-C₈ cycloalkyl, C₁-C₈ alkyl, C₆-C₁₄aryl, substituted C₆-C₁₄ aryl, C₁-C₁₄ heteroaryl, substituted C₁-C₁₄heteroaryl, non-aryl C₁-C₁₄ heterocycle or substituted non-aryl C₁-C₁₄heterocycle. As used herein, “C₁-C₈ alkyl” refers to a saturatedhydrocarbon which may be straight chained or branched. Examples aremethyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl. “C₆-C₁₄aryl” and “C₁-C₁₄ heteroaryl” are as defined above. Examples areprovided above. A “substituted C₆-C₁₄ aryl” is a C₆-C₁₄ aryl wherein atleast one ring hydrogen is replaced with one or more atoms other thanhydrogen. A “substituted C₁-C₁₄ heteroaryl” is a C₁-C₁₄ heteroarylwherein at least one ring hydrogen or hydrogen attached to a heteroatomis replaced with one or more atoms other than hydrogen. Such atomsinclude carbon, oxygen, nitrogen, sulfur and halogen. A “non-aryl C₁-C₁₄heterocycle” refers to a non-aromatic substituent with one to fourteencarbon atoms (with at least one heteroatom) in one or multiple ringswhich may be separated by a bond or fused. The third atom of a threemember ring may be provided by the carbon to which R³ is attached.Examples include piperidine, piperazine, pyrrolidine, and their oxygenand sulfur equivalents. A “substituted non-aryl C₁-C₁₄ heterocycle” is anon-aryl C₁-C₁₄ heterocycle wherein at least one ring hydrogen orhydrogen attached to a heteroatom is replaced with one or more atomsother than hydrogen. Such atoms include carbon, oxygen, nitrogen, sulfurand halogen. Examples of R³ include NHCOPh, NHCO—C₆H₄—Cl (para),NHCO—C₆H₄—OMe (para), NHCO—C₆H₄—NO₂ (para), NHCO—C₆H₄-Ph (para),NHCO—C₆H₃(OMe)₂ (meta, para), NHCO(2-naphthyl), NHCO—C₆H₄—OCH₂Ph (para),N(CH₂Ph)COPh, NHCOCH₂CH₂Ph, NHCOCHPh₂, NHCOMe, NHCO(cyclo-C₆H₁₁),NHSO₂—C₆H₄-Me (para), NHCONHEt, NHCONHPh, NHCOOCH₂—C₆H₄—NO₂ (para),NHCOOCH₂(2-naphthyl), or NHCOOCH₂Ph. The abbreviation “Ph” represents“phenyl”.

Examples of R⁴ substituents include H, NHAC, and NHAC substituted withN-glycolyl. It also includes —O-Lactose, substituted —O-Lactose,—O-Lactosamine, substituted —O-Lactosamine, polyethylene glycol andsubstituted polyethylene glycol.

Examples of R⁵ substituents include H, NHAc, and NHAc substituted withN-glycolyl.

Examples of non-monosaccharide, ringed substituents include:

For certain embodiments, it may be beneficial to also, or alternatively,link a diagnostic or therapeutic agent, such as a drug to a Glycomimeticcompound, to form a conjugate where the linkage is covalent. As usedherein, the term “therapeutic agent” refers to any bioactive agentintended for administration to a warm-blooded animal (e.g., a mammalsuch as a human) to prevent or treat a disease or other undesirablecondition or to enhance the success of therapies. Therapeutic agentsinclude antibiotics, hormones, growth factors, proteins, peptides,genes, non-viral vectors and other compounds.

Glycomimetic Compound Formulations

Glycomimetic compounds as described herein may be present within apharmaceutical composition. A pharmaceutical composition comprises oneor more Glycomimetic compounds in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers (e.g., neutralbuffered saline or phosphate buffered saline), carbohydrates (e.g.,glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/orpreservatives. Within yet other embodiments, compositions of the presentinvention may be formulated as a lyophilizate. Compositions of thepresent invention may be formulated for any appropriate manner ofadministration, including for example, aerosol, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration.

A pharmaceutical composition may also, or alternatively, contain one ormore active agents, such as drugs (e.g., antibiotics), which may belinked to a Glycomimetic compound or may be free within the composition.The attachment of an agent to a Glycomimetic compound may be covalent ornoncovalent. An example of an active agent is tobramycin. Tobramycinalone has typically been administered intravenously or by inhalation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Carriers for use within such formulations are biocompatible, andmay also be biodegradable; preferably the formulation provides arelatively constant level of modulating agent release. The amount ofGlycomimetic compound contained within a sustained release formulationdepends upon the site of implantation, the rate and expected duration ofrelease and the nature of the condition to be treated or prevented.

Glycomimetic compounds are generally present within a pharmaceuticalcomposition in a therapeutically effective amount. A therapeuticallyeffective amount is an amount that results in a discernible patientbenefit, such as a measured or observed response of a conditionassociated with Pseudomonas infection.

Glycomimetic Compounds Methods of Use

In general, Glycomimetic compounds described herein may be used forachieving diagnostic and/or therapeutic results in disease (e.g., humandisease) involving infection by Pseudomonas (e.g., P. aeruginosa)bacteria. Such diagnostic and/or therapeutic results may be achieved invitro and/or in vivo in an animal, preferably in a mammal such as ahuman, provided that Pseudomonas (e.g., P. aeruginosa) or its productsare ultimately contacted with a Glycomimetic compound, in an amount andfor a time sufficient to achieve a discernable diagnostic or therapeuticresult. In the context of this invention, a therapeutic result wouldrelate, for example, to the prevention of lung infections. In someconditions, therapeutic results would be associated with the inhibitingof Pseudomonas (such as P. aeruginosa) or its products (where inhibitingincludes, for example, arresting the growth of or killing the bacteriaor preventing colonization by the bacteria). As used herein, therapy ortherapeutic results includes treatment or prevention.

Glycomimetic compounds of the present invention may be administered in amanner appropriate to the disease to be treated or prevented.Appropriate dosages and a suitable duration and frequency ofadministration may be determined by such factors as the condition of thepatient, the type and severity of the patient's disease and the methodof administration. In general, an appropriate dosage and treatmentregimen provides the modulating agent(s) in an amount sufficient toprovide treatment and/or prophylactic benefit. Within particularlypreferred embodiments of the invention, a Glycomimetic compound may beadministered at a dosage ranging from 0.001 to 1000 mg/kg body weight(more typically 0.01 to 1000 mg/kg), on a regimen of single or multipledaily doses. Appropriate dosages may generally be determined usingexperimental models and/or clinical trials. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art.Glycomimetic compounds described herein may be administered incombination (i.e., simultaneously or sequentially) with anotheranti-bacterial compound. For example, a Glycomimetic compound may beadministered in combination with tobramycin.

Glycomimetic compounds may also be used to target substances toPseudomonas bacteria , e.g., P. aeruginosa. Such substances includetherapeutic agents and diagnostic agents. Therapeutic agents may be amolecule, virus, viral component, cell, cell component or any othersubstance that can be demonstrated to modify the properties of a targetcell so as to provide a benefit for treating or preventing a disorder orregulating the physiology of a patient. A therapeutic agent may also bea drug or a prodrug that generates an agent having a biological activityin vivo. Molecules that may be therapeutic agents may be, for example,polypeptides, amino acids, nucleic acids, polynucleotides, nucleosides,steroids, polysaccharides or inorganic compounds. Such molecules mayfunction in any of a variety of ways, including as enzymes, enzymeinhibitors, hormones, receptors, antisense oligonucleotides, catalyticpolynucleotides, anti-viral agents, anti-tumor agents, anti-bacterialagents, immunomodulating agents and cytotoxic agents (e.g.,radionuclides such as iodine, bromine, lead, rhenium, homium, palladiumor copper). Diagnostic agents include imaging agents such as metals andradioactive agents (e.g., gallium, technetium, indium, strontium,iodine, barium, bromine and phosphorus-containing compounds), contrastagents, dyes (e.g., fluorescent dyes and chromophores) and enzymes thatcatalyze a calorimetric or fluorometric reaction. In general,therapeutic and diagnostic agents may be attached to a Glycomimeticcompound using a variety of techniques such as those described above.For targeting purposes, a Glycomimetic compound may be administered to apatient as described herein.

Glycomimetic compounds may also be used in vitro, e.g., within a varietyof well known cell culture and cell separation methods. For example, aGlycomimetic compound may be immobilized on a solid support (such aslinked to the interior surface of a tissue culture plate or other cellculture support) for use in immobilizing Pseudomonas bacteria or theirproducts for screens, assays and growth in culture. Such linkage may beperformed by any suitable technique, such as the methods describedabove, as well as other standard techniques. Glycomimetic compounds mayalso be used to facilitate cell identification and sorting in vitro,permitting the selection of such bacterial cells. Preferably, theGlycomimetic compound(s) for use in such methods is linked to adiagnostic agent which is a detectable marker. Suitable markers are wellknown in the art and include radionuclides, luminescent groups,fluorescent groups, enzymes, dyes, constant immunoglobulin domains andbiotin. Within one preferred embodiment, a Glycomimetic compound linkedto a fluorescent marker, such as fluorescein, is contacted with thecells, which are then analyzed by fluorescence activated cell sorting(FACS).

Such in vitro methods generally comprise contacting a sample (e.g., abiological preparation) with any one of the Glycomimetic compounds, anddetecting the compound in the sample. If desired, one or more wash stepsmay be added to a method. For example, subsequent to contacting a samplewith a Glycomimetic compound but prior to detection of the compound, thesample may be washed (i.e., contacted with a fluid and then removal ofthe fluid in order to remove unbound Glycomimetic compound).Alternatively, or in addition, a wash step may be added during thedetection process. For example, if a Glycomimetic compound possesses amarker (a diagnostic agent) that can bind to a substance that isdetectable, it may be desirable to wash the sample subsequent tocontacting the sample with a detectable substance, but prior to thedetection. As used herein, the phrase “detecting the compound (or agent)in the sample” includes detecting the compound (or agent) while it isbound to the sample, or detecting the compound (or agent) which wasbound to the sample but after it has been separated from the sample.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Synthesis of Glycomimetic 1

Reagents and solvents were used as received from commercial suppliers.Thin-layer chromatography (TLC) was performed using Analtech silica gelplates and visualized by UV light (254 nm) stain. Progress of thereactions was monitored by TLC or GC.

Preparation of B4 Derivatives

Preparation of B4-2

A solution of B4-1 (150 g, 385 mmol) in CH₂Cl₂ (800 mL) was added slowlyto a slurry of ZrCl₄ (85 g, 366 mmol) in CH₂Cl₂ (800 mL) at 0° C. Afterthe reaction was stirred at 0° C. for 30 min, EtSH (32 mL, 423 mmol) wasadded and the reaction was complete within 2 h. The reaction wasquenched by brine (500 mL). The layers were separated and the organiclayer was washed with saturated aqueous NaHCO₃ (1×500 mL) and brine(1×500 mL), dried over MgSO₄, filtered, and concentrated to give an oilyresidue. This was dissolved in MTBE (300 mL) and the solution wasdiluted with heptane (1.2 L). After the resulting slurry had beenstirred in an ice bath for 1 h, the mixture was filtered to providecompound B4-2 (143 g, 95%).

Preparation of B4

NaOMe (0.5 M in MeOH, 1.09 L, 545 mmol) was added to a solution of B4-2(710 g, 392 mmol) in MeOH (4 L) at room temperature. After 2 h, Dowex50wx8-200 resin (350 g) was added until the pH reached 3.5-4. The resinwas filtered off and the filtrate was concentrated to an oily residue.This was dissolved in CH₂Cl₂ and concentrated again to give B4 as asolid (367 g, 90%).

Preparation of B4-3

A solution of B4 (10 g, 44.6 mmol) in anhydrous DMF (125 mL) was addedto a suspension of NaH (14 g, 357 mmol, 60% in mineral oil) in anhydrousDMF (125 mL) at 0° C. After the reaction was stirred at room temperaturefor 30 min and then cooled to 0° C., BnBr (42 mL, 357 mmol) was addedcautiously. The reaction was stirred at room temperature for 2 h andthen cautiously quenched with MeOH (30 mL) while cooling in an ice bath.Water (200 mL) was added and the mixture was extracted with EtOAc (2×200mL). The organic phase was washed with water (2×500 mL) and brine, driedover MgSO₄, and concentrated to an oily residue. Chromatographicpurification on silica gel eluted with EtOAc/heptane (1:9) provided B4(20.6 g, 79%).

Preparation of B2 Derivatives

Preparation of B2-2

Triethylamine (6.8 L, 48.7 mol) was added to a solution of B2-1 (1 kg,6.1 mol) and DMAP (74 g, 0.61 mol) in CH₂Cl₂ (12 L), followed byaddition of Ac₂O (3.46 L, 36.6 mol) at 0-10° C. The reaction was stirredat room temperature oversight and then quenched with water (25 L). Thelayers were separated. The organic phase was washed with water (25 L),saturated aqueous NaHCO₃ (2×10 L), and brine (10 L), dried over MgSO₄,and concentrated to provide crude B2-2 (2.1 kg, >quantitative).

Preparation of B2-3

A solution of B2-2 (1.5 kg, 3.01 mol) in CH₂Cl₂ (6 L) was added slowlyto a slurry of ZrCl₄ (1 kg, 2.86 mol) in CH₂Cl₂ (1.2 L) at 0-5° C. Afterthe reaction was stirred for 30 min, EtSH (351 mL, 3.16 mol) was added.The reaction was stirred at 0° C. Due to incomplete reaction, additionalEtSH (3×3051 mL) was added during the first 30 h of the reaction. After2 days the mixture was quenched with brine (7.5 L). The layers wereseparated and the organic layer was washed with saturated NaHCO₃ (6 L)and brine (6 L), dried over MgSO₄, and concentrated to give an oilyresidue. The residue was recrystallized from MTBE/heptane (1:3) to givea first crop of B2-3. The mother liquors were concentrated and theresidue was purified by chromatography on silica gel eluted withEtOAc/heptane (2:8) to give a second crop. A total of 560 g of B2-3 (38%over two steps) was obtained.

Preparation of B2-4

NaOMe (25 wt % in MeOH, 119 mL, 519 mmol) was added slowly into asolution of B2-3 (579 g, 1.73 mol) in MeOH (2.3 L) at room temperature.After 1 h, Dowex 50wx8-200 resin (290 g) was added and the reaction wasstirred for 30 min. The resin was filtered off and the filtrate wasconcentrated to afford solid B2-4 (360 g, quantitative).

Preparation of B2

A solution of B2-4 (360 g, 1.73 mol) in THF (3.5 L) was treated withpotassium tert-butoxide (20 wt % in THF, 3.88 kg, 6.91 mol) at 0° C.After 30 min, BnBr (822 mL, 6.91 mol) was added. The reaction wasstirred at room temperature for 2 h and then cooled to 10° C. Afterovernight stirring, the reaction was quenched with saturated aqueousNH₄Cl (2 L) and then filtered through a pad of Celite. The filtrate wasextracted with EtOAc (5.5 L). The organic phase was washed with brine (4L) and concentrated. Chromatographic purification eluted withEtOAc/heptane (1:9) provided B2 (460 g, 56%).

Preparation of B5 Derivatives

Preparation of B5-2

A suspension of B5-1 (184 g, 676 mmol, Aldrich) and Pd/C (10 wt %, 50%water wet; 19 g) in THF (800 mL) was hydrogenated at 50 psi overnight atroom temperature. The catalyst was filtered off through a pad of Celite,the filter pad was washed with ethyl acetate, and the combined filtratewas concentrated. The residue was dissolved in ethyl acetate and thesolution was then washed with water (250 mL), aqueous sodium bicarbonate(250 mL) and then brine (250 mL), dried over MgSO₄ and concentrated togive B5-2 as an oily residue (217 g, quantitative).

Preparation of B5-3

NaOMe (25 wt % in MeOH, 236 mL, 1.03 mol) was added slowly into asolution of B5-2 (189 g, 689 mmol) in MeOH (600 mL) at room temperature.After 2 h, Dowex 50wx8-200 (320 g) was added to adjust pH to 3.5-4. Theresin was filtered off and the filtrate was concentrated. The residuewas azeotroped with toluene and then with acetonitrile to give B5-3 (102g, quantitative) as a waxy solid.

Preparation of B5

A suspension of B5-3 (69 g, 465 mmol) in CH₃CN (1.2 L) was treated withbenzaldehyde dimethyl acetal (76.9 mL, 512 mmol) and a solution ofp-toluenesulfonic acid monohydrate (4.56 g, 24 mmol) in CH₃CN (75 mL).After stirring at room temperature for 1 h, the reaction was heated toreflux for 1 h and then cooled to room temperature and neutralized withEt₃N (3 mL). The mixture was concentrated and the residue was dissolvedwith EtOAc (800 mL). The solution was washed with water (500 mL) andthen brine (500 mL), dried over MgSO₄, and concentrated to give a crudeproduct. This was dissolved in ethyl acetate (100 mL) at 70° C. and thentreated with heptane (270 mL, in 50 mL portions). The mixture was cooledto room temperature and filtered to give a first crop of B5.Chromatographic purification of the mother liquors on silica gel elutedwith EtOAc/heptane (3:7) gave a second crop. A total of 88 g of B5 (8%)was obtained.

Preparation of Glycomimetic 1

Preparation of Compound 1

A suspension of B5 (20 g, 87.4 mmol), B4-2 (40 g, 101.6 mmol), andN-iodosuccinimide (25 g, 110.1 mmol) in CH₂Cl₂ (230 mL, anhydrous) wastreated with trifluoromethanesulfonic acid (0.15 M in CH₂Cl₂, about 2mL; freshly prepared before use) at 0° C. until the color changed fromlight red brown to dark brown. The reaction was stirred in an ice bathfor 40 min and then quenched with aqueous Na₂CO₃ (8%, 100 mL) to pH 9.After dilution with CH₂Cl₂ (100 mL) and water (100 mL), the layers wereseparated. The organic phase was washed with aqueous Na₂S₂O₃ (10%, 370mL) and then brine (100 mL), dried over Na₂SO₄, and concentrated.Chromatographic purification on silica gel eluted with MTBE/heptane(6:4) gave 1 (40.8 g, 81%) as a foam.

Preparation of Compound 2

A solution of NaBH₃CN (42 g, 670 mmol) in anhydrous THF (280 mL) wasadded into a solution of compound 1 (38 g, 67 mmol) in anhydrous THF (70mL) at room temperature. Molecular sieves (3 Å, powder, dried; 30 g)were then added and the reaction mixture was stirred at room temperaturefor 30 min before being cooled to 0° C. HCl (in anhydrous Et₂O, preparedbefore use; ˜90 mL) was added slowly until the complete consumption byTLC of compound 1. Solid Na₂CO₃ (35 g) was added portionwise into thereaction with cooling of an ice bath and the slurry was stirred for 10min before filtering through a pad of Celite. The filtrate was dilutedwith EtOAc (300 mL), washed with saturated aqueous NaHCO₃ (100 mL) andbrine (100 mL), and concentrated to a yellow oil. This residue waspartitioned between toluene (400 mL) and water (200 mL) but three layersformed. The layers were separated and the middle layer was repeatedlypartitioned between toluene (6×400 mL) and water (6×100 mL). Thecombined organic layers were concentrated to give crude 2 (35 g).Chromatographic purification on silica gel eluted with EtOAc/heptane(6:4) provided 2 (26 g, 68%) as an oily foam.

Preparation of Compound 3

Part 1: Br₂ (distilled over P₂O₅, 1.8 mL, 34.8 mmol) was added dropwiseto a solution of B2 (13.9 g, 29 mmol) in anhydrous CH₂Cl₂ (30 mL) at 0°C. After the reaction had been stirred in an ice bath for 40 min,cyclohexene (4 mL) was added until the color changed to a persistentyellow.

Part 2: Compound 2 (11 g, 19.4 mmol) was dissolved in anhydrous CH₂Cl₂(80 mL), followed by addition of Et₄NBr (dried at 200° C. for 2 h underN₂; 6.1 g, 29 mmol), anhydrous DMF (50 mL), and molecular sieves (4 Åpowder, dried; 12 g). After the reaction mixture had been stirred atroom temperature for 30 min, the solution from part 1 was added. Thereaction mixture was stirred for 40 h at room temperature and thendiluted with EtOAc (100 mL) and filtered through a pad of Celite. Thefiltrate was washed with aqueous Na₂S₂O₃ (10%, 100 mL), water (100 mL),and then brine (100 mL), dried over Na₂SO₄, and concentrated.Chromatographic purification on silica gel eluted with EtOAc/heptane(1:1) gave 3 (15 g, 78%) as an oily foam.

Preparation of Compound 4

NaOMe (0.5 M in MeOH, 4.6 mL, 2.24 mmol) was added to a solution ofcompound 3 (11 g, 11.2 mmol) in MeOH (30 mL) at room temperature. After3 h, AcOH (1 mL) was added and pH reached 4. The mixture wasconcentrated to give crude 4 (9.2 g, quantitative), used withoutadditional purification in the next step.

Preparation of Compound 5

A mixture of compound 4 (9.2 g, 12.3 mmol) and dibutyltin oxide (3.92 g,15.8 mmol) in anhydrous MeOH (150 mL) was heated to reflux for 4 h andthen the reaction was evaporated to dryness. The residue was dissolvedin toluene (150 mL, anhydrous) in a dry flask. Tetrabutylammoniumbromide (3.65 g, 7.91 mmol) and allyl bromide (1.6 mL, 18.1 mmol) wereadded. The reaction mixture was heated to reflux for 4 h, cooled andthen concentrated. The residue was purified by chromatography on silicagel eluted with EtOAc/CH₂Cl₂ (7:3) to give 5 (6.4 g, 67% over twosteps).

Preparation of Compound 6

A solution of compound 5 (6.3 g, 7.35 mmol) in anhydrous DMF (30 mL) wasadded into a suspension of NaH (1.47 g, 36.8 mmol, 60% in mineral oil)in anhydrous DMF (30 mL) at 0° C. After the reaction had been stirred atroom temperature for 30 min, it was cooled to 0° C. and BnBr (3.95 mL,33.1 mmol) was added. The reaction was stirred at room temperature for 2h and then quenched with MeOH (1.5 mL) in an ice bath. Water (20 mL) andice cold aqueous HCl (1 M, 100 mL) were added, followed by extractionwith CH₂Cl₂ (200 mL). The layers were separated and the organic phasewas washed with water (200 mL), ice cold saturated aqueous NaHCO₃ (100mL), and brine, dried over Na₂SO₄, and concentrated. Chromatographicpurification on silica gel eluted with EtOAc/heptane (1:4) provided 6(6.36 g, 78%).

Preparation of Compound 7

KOtBu (1.57 g, 14.0 mmol) was added to a solution of compound 6 (6.3 g,5.59 mmol) in DMSO (40 mL) at room temperature. The slurry was heated to100° C. in a pre-heated oil bath for 2 h. After the reaction had beencooled to room temperature, CH₂Cl₂ (150 ML) and water (100 mL) wereadded. The layers were separated and the aqueous phase was extractedwith CH₂Cl₂ (2×150 mL). The combined organics were washed with water(3×100 mL) and then brine (100 mL), dried over Na₂SO₄, and concentratedto a residue (6.4 g). This was dissolved with acetone (40 mL) and water(4 mL), HgO (yellow; 3.3 g, 15.1 mmol) was added followed by a solutionof HgCl₂ (anhydrous; 3.3 g, 12.3 mmol) in acetone (40 mL) and water (4mL) dropwise. After 30 min, the mixture was filtered through a pad ofCelite and the filtrate was concentrated. The residue was partitionedbetween CH₂Cl₂ (200 mL) and saturated aqueous NaI (30 mL). The organiclayer was washed with water (100 mL) and then brine, dried over Na₂SO₄,and concentrated. Chromatographic purification on silica gel eluted withEtOAc/heptane (1:4) provided 7 (4.35 g, 720/%) as a yellow oil.

Preparation of Compound 8

Part 1: Br₂ (distilled over P₂O₅, 0.39 mL, 7.59 mmol) was added dropwiseto a solution of B4-3 (4.03 g, 6.9 mmol) in anhydrous CH₂Cl₂ (10 mL) at0° C. After the reaction had been stirred in an ice bath for 40 min,cyclohexene (1 mL) was added until the color changed to a persistentyellow.

Part 2: Compound 7 (3.75 g, 3.45 mmol) was dissolved in anhydrous CH₂Cl₂(25 mL) and then Et₄NBr (dried at 200° C. for 2 h under N₂, 1.45 g, 6.9mmol), anhydrous DMF (15 mL), and molecular sieves (4 Å powder, dried; 4g) were added. After this mixture had been stirred at room temperaturefor 30 min, the solution from part 1 was added. The reaction was stirredfor 60 h at room temperature and then diluted with EtOAc (50 mL) andfiltered through a pad of Celite. The filtrate was washed with aqueousNa₂S₂O₃ (10%, 50 mL), water (30 mL), and then brine (30 mL), dried overNa₂SO₄, and concentrated. Chromatographic purification on silica geleluted with EtOAc/heptane (1:3) gave 8 (3.94 g, 71%) as a foam.

Preparation of Glycomimetic 1

A suspension of 8 (3.9 g, 2.42 mmol) and Pd(OH)₂/C (20 wt %, 50% waterwet; 2 g) in MeOH (150 mL), 1,4-dioxane (15 mL), and AcOH (5 mL) washydrogenated under 60 psi at room temperature for 20 h. The mixture wasfiltered through a pad of Celite and the filtrate was concentrated.Chromatographic purification on silica gel eluted with CH₂Cl₂/MeOH/H₂O(10:9:1) gave Glycomimetic 1 (1.05 g, 70%) as a white solid.

Example 2 Synthesis of Glycomimetic (Compound XXVIII)

Chemical structures are depicted in FIGS. 3A-3B.

Synthesis of intermediate XVIV: To a solution of compound XVIII (0.8 g)in acetonitrile (10 ml) is added benzaldehydedimethylacetal (0.5 g) andp-toluenesulfonic acid (0.2 mg) and the mixture is stirred at roomtemperature for 6 h. Triethylamine (0.2 ml) is added and the reactionmixture is stirred at room temperature for 5 min. Solvent is evaporatedoff and the residue is purified by column chromatography (silica gel) togive intermediate XVIV.

Synthesis of intermediate XXV: To a solution of compound XVIV (0.5 g) inDMF (5 ml) is added sodium hydride (0.1 g, 600/% suspension in oil) withstirring. Benzyl bromide (0.2 ml) is added drop wise to the abovereaction mixture and stirred at room temperature for 16 h. Methanol (0.5ml) is added with stirring and the reaction mixture is concentrated todryness. Dichloromethane (50 ml) is added and the organic layer iswashed successively with cold 1N HCl cold sodium bicarbonate solutionand cold water. Organic layer is dried (sodium sulfate), filtered andconcentrated to dryness. Residue is purified by column chromatography(silica gel) to give compound XXV.

Synthesis of intermediate XXVI: To a cold solution of XXV (0.4 g) in THF(5 ml) is added sodiumcyanoborohydride (0.1 g). A cold solution of HClin ether is added drop wise until effervescence ceases (pH 2-3).Reaction mixture is diluted with ether (50 ml) and washed successivelywith a cold aqueous solution of sodium bicarbonate, and cold water.Solvent is evaporated off and the residue is purified by columnchromatography (silica gel) to give compound XXVI.

Synthesis of intermediate XXVII: To a solution of compound XXVI (0.1 g)in dichloromethane-DMF (2 ml) is added molecular sieves (4 A, 0.1 g) andtetraethyl ammonium bromide (0.05 g) and the reaction mixture is stirredfor 1 h at room temperature under argon. To this reaction mixture isadded compound XVI (0.1 g) and the reaction mixture is stirred at roomtemperature for 48 h under argon. Reaction mixture is diluted withdichloromethane (10 ml) and washed successively with cold saturatedsodium bicarbonate solution and water; then dried (sodium sulfate),filtered and concentrated to dryness. The residue is purified by columnchromatography (silica gel) to give compound XXVII.

Synthesis of compound XXVIII: To a solution of compound XXVII (0.06 g)in dioxane-water (5 ml, 6:1) is added acetic acid (10 drops) and 10%Pd—C (0.06 g). The reaction mixture is vigorously shaken under hydrogenfor 20 h. Reaction mixture is filtered through a bed of celite and thesolvent is evaporated off. Residue is purified by passing through acolumn of sephadex G-10 to give compound XXVIII.

Example 3 Synthesis of Glycomimetic (Compound XXXIII)

Chemical structures are depicted in FIGS. 3A-3B.

Synthesis of intermediate XXIX: To a solution of compound XVIII (2 g) inpyridine (20 ml) is added tert-butyl-dimethylsialylchloride (0.6 g) andthe solution is stirred at room temperature for 16 h. Solvent isevaporated off and the residue is purified by column chromatography(silica gel) to give compound XXIX.

Synthesis of intermediate XXX: To a suspension of compound XXIX (1 g) inα,α-dimethoxy propane (10 ml) is added camphorsulfonic acid (0.2 g) andthe reaction mixture is stirred at room temperature for 16 h.Triethylamine (0.2 ml) is added and the solvent is evaporated off. Theresidue is purified by column chromatography (silica gel) to givecompound XXX.

Synthesis of intermediate XXXI: Compound XXX (0.8 g) is reacted withcompound XVI (0.8 g) in the same way as described in Example 2 to givecompound XXXI.

Synthesis of intermediate XXXII: To a solution of compound XXXI (0.5 g)in acotonitrile (5 ml) is added triethylamine (0.1 ml) and a solution ofH₂SiF₆ (0.5 ml, 35%) in acetonitrile (1 ml). After 2 h, the reactionmixture is diluted with dichloromethane (50 ml) and washed with coldsaturated solution of sodium bicarbonate and cold water, then dried(sodium sulfate), filtered, and concentrated to dryness. The residue ispurified by column chromatography (silica gel) to give compound XXXII.

Synthesis of compound XXXIII: A solution of compound XXXII (0.2 g) in60% acetic acid in water is heated at 60° C. for 1 h. Solvent isevaporated off and the crude product is dissolved in dioxan-water (5 ml,6:1). Acetic acid (10 drops) is added followed by 10% Pd—C. Thesuspension is shaken under hydrogen for 24 h, filtered (Celte bed) andconcentrated to dryness. The residue is purified by passing through acolumn of sephadex G-10 to give compound XXXIII.

Example 4 Synthesis of Glycomimetic (Compound XXXVII)

Chemical structures are depicted in FIGS. 3A-3B.

Synthesis of intermediate XXXIV: A solution of XXIX (1 g) in DMF istreated with NaH (0.14 g) and benzyl bromide (0.4 ml) in same way asdescribed in Example 2 and purified by column chromatography (silicagel) to give compound XXXIV.

Synthesis of intermediate XXXV: Compound XXXIV (1 g) is treated withH₂SiF₆ in the same way as described in Example 3 to give compound XXXV.

Synthesis of intermediate XXXVI: Intermediate XXXV (0.5 g) is reactedwith intermediate XVI (0.4 g) as described in Example 2 to give compoundXXXVI.

Synthesis of compound XXXVII: Intermediate XXXVI (0.3 g) is hydrogenatedas described in Example 2 and purified by sephadex G-10 to give compoundXXXVII.

Example 5 Assay for PA-IL Antagonist Activity

Wells of a microtiter plate (plate 1) are coated with PA-IL(Sigma-Aldrich, St. Louis, Mo.) by incubation for 2 hrs at 37° C. Thewells are then blocked for 2 hrs by the addition of 1% bovine serumalbumin (BSA) diluted in TBS-Ca (50 mM TrisHCl, 150 mM NaCl, 2 mM CaCl₂pH 7.4) mixed 1:1 v/v with Stabilcoat (Surmodics, Eden Prairie, Minn.).In a second low-binding round-bottom microtiter plate (plate 2), testantagonists are serial diluted in 1% BSA in TBS-Ca/Stabilcoat (60μl/well). Preformed conjugates of α-galactose-PAA-biotin (GlycoTechCorp, Gaithersburg, Md.) mixed with streptavidin-HRP (KPL Labs,Gaithersburg, Md.) are added to each well of plate 2 (60 μl/well of 2μg/ml). Plate 1 is then washed with TBS-Ca and 100 μl/well aretransferred from plate 2 to plate 1. After incubation at roomtemperature for 2 hrs, plate 1 is washed and 100 μl of TMB reagent (KPLLabs, Gaithersburg, Md.) is added to each well. After incubation for 5minutes at room temperature, the reaction is stopped by adding 100μl/well of 1M H₃PO₄ and the absorbance of light at 450 nm is determinedby a microtiter plate reader.

Example 6 Assay for PA-IIL Antagonist Activity

Wells of a microtiter plate (plate 1) are coated with PA-IIL (Dr.Wimmerova, Masaryk University, Brno, Czech Republic) by incubation for 2hrs at 37° C. The wells are then blocked for 2 hrs by the addition of 1%bovine serum albumin (BSA) diluted in TBS-Ca (50 mM TrisHCl, 150 mMNaCl, 2 mM CaCl₂ pH 7.4) mixed 1:1 v/v with Stabilcoat (Surmodics, EdenPrairie, Minn.). In a second low-binding round-bottom microtiter plate(plate 2), test antagonists are serial diluted in 1% BSA inTBS-Ca/Stabilcoat (60 μl/well). Preformed conjugates offucose-PAA-biotin (GlycoTech Corp, Gaithersburg, Md.) mixed withstreptavidin-HRP (KPL Labs, Gaithersburg, Md.) are added to each well ofplate 2 (60 μl/well of 2 μg/ml). Plate 1 is then washed with TBS-Ca and100 μl/well are transferred from plate 2 to plate 1. After incubation atroom temperature for 2 hrs, plate 1 is washed and 100 μl of TMB reagent(KPL Labs, Gaithersburg, Md.) is added to each well. After incubationfor 5 minutes at room temperature, the reaction is stopped by adding 100μl/well of 1M H₃PO₄ and the absorbance of light at 450 nm is determinedby a microtiter plate reader.

Example 7 Assay for Inhibition of PA-I or PA-II Lectin Binding to BuccalCells

Obtain sample of buccal cells by scraping inside of cheek and collectingin 2 mis PBS. Spin cells at 400 g for 7 minutes to generate cell pellet.Discard supernatant. Resuspend in cold TBS-Ca (50 mM TrisHCl, 150 mMNaCl, 2 mM CaCl₂ pH 7.4) to cell concentration of 10⁶ cells/ml. Aliquot0.1 ml to each tube. Add biotinylated PA-I or PA-II to tubes (5μ/well of1.0 mg/ml lectin). Add inhibitors to tubes (5 μl at desiredconcentration). Incubate on ice for 30 minutes. Wash cells once byadding 400 μl of cold TBS-Ca to each tube and spinning at 400 g for 7minutes. Discard supernatant. Resuspend cells in 100 μl of cold TBS-Ca.Add streptavidin-FITC (2 μl/tube of 1 mg/ml, KPL Labs, Gaithersburg,Md.). Incubate 30 minutes on ice. Wash cells once by adding 400 μl ofcold TBS-Ca to each tube and spinning at 400 g for 7 minutes. Discardsupernatant. Resuspend cells in 500 μl of cold TBS-Ca. Analyze in flowcytometer.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A compound or physiologically acceptable salt thereof, having theformula:

wherein: where —O— separating the two rings in the formula is in analpha or beta 1-3 linkage; R^(1a), R^(1b), R^(1c) and R^(1d) are eachindependently selected from OH, NHAc, and galactose linked by an Oglycosidic bond, with the proviso that three of R^(1a), R^(1b), R^(1c)and R^(1d) are independently selected from OH and NHAc and one ofR^(1a), R^(1b), R^(1c) and R^(1d) is not OH or NHAc; R²=a fucose;R³=—CH₂—OH; R⁴=H; and R⁵=H.
 2. The compound or salt thereof according toclaim 1 wherein R^(1a) is not OH or NHAc.
 3. The compound or saltthereof according to claim 1 wherein R^(1c) is not OH or NHAc.
 4. Thecompound or salt thereof according to claim 1 wherein R^(1d) is not OHor NHAc.
 5. The compound or salt thereof according to claim 1, where —O—separating the two rings in the formula is in a beta 1-3 linkage.
 6. Thecompound or salt thereof according to claims 1-2, 3-4 or 5 wherein thecompound or salt thereof is in combination with a pharmaceuticallyacceptable carrier or diluent.